Receptor-mediated Endocytosis Is Not Required for Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 °C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 °C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells. Induction of apoptosis occurs primarily by two main pathways, the Bcl-2 family regulated pathway (also known as the intrinsic or mitochondrial pathway) and the extrinsic pathway (1Ashkenazi A. Nat. Rev. Cancer. 2002; 2: 420-430Crossref PubMed Scopus (1099) Google Scholar, 2Bratton S.B. MacFarlane M. Cain K. Cohen G.M. Exp. Cell Res. 2000; 256: 27-33Crossref PubMed Scopus (288) Google Scholar, 3Reed J.C. Am. J. Pathol. 2000; 157: 1415-1430Abstract Full Text Full Text PDF PubMed Google Scholar, 4Strasser A. Nat. Rev. Immunol. 2005; 5: 189-200Crossref PubMed Scopus (514) Google Scholar). Caspase-9, the apical caspase in the Bcl-2 family regulated pathway, is activated after perturbation of mitochondria resulting from cellular stress, growth factor withdrawal, or cytotoxic insults. Caspase-8 is the apical caspase in the extrinsic pathway and is activated after ligation of death receptors by members of the TNF 3The abbreviations used are: TNF, tumor necrosis factor; TNF-R, TNF receptor; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R, TRAIL receptor; DR, death receptor; FADD, Fas-associated death domain; DISC, death-inducing signaling complex; TMRE, tetramethylrhodamine ethyl ester; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; WT, wild type; RIP, receptor-interacting protein. 3The abbreviations used are: TNF, tumor necrosis factor; TNF-R, TNF receptor; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R, TRAIL receptor; DR, death receptor; FADD, Fas-associated death domain; DISC, death-inducing signaling complex; TMRE, tetramethylrhodamine ethyl ester; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; WT, wild type; RIP, receptor-interacting protein. family, including TNF, CD95L (FasL/Apo-1L), and TNF-related apoptosis-inducing ligand (TRAIL). Ligation of cell surface death receptors, such as CD95, TNF receptor 1 (TNF-R1), and TRAIL-R1 (DR4) or TRAIL-R2 (DR5), by their cognate ligands or agonistic antibodies, results in receptor aggregation and recruitment of the adaptor protein MORT1/FADD (Fas-associated death domain) (1Ashkenazi A. Nat. Rev. Cancer. 2002; 2: 420-430Crossref PubMed Scopus (1099) Google Scholar, 2Bratton S.B. MacFarlane M. Cain K. Cohen G.M. Exp. Cell Res. 2000; 256: 27-33Crossref PubMed Scopus (288) Google Scholar, 4Strasser A. Nat. Rev. Immunol. 2005; 5: 189-200Crossref PubMed Scopus (514) Google Scholar, 5Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Crossref PubMed Scopus (1123) Google Scholar). FADD then recruits the initiator caspase-8, which is activated within the death-inducing signaling complex (DISC) (6Kischkel F.C. Hellbardt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1771) Google Scholar, 7Peter M.E. Krammer P.H. Cell Death Differ. 2003; 10: 26-35Crossref PubMed Scopus (891) Google Scholar). Currently there is much interest in exploring the therapeutic potential of TRAIL because it induces apoptosis selectively in many cancer cells but not in most normal cells (1Ashkenazi A. Nat. Rev. Cancer. 2002; 2: 420-430Crossref PubMed Scopus (1099) Google Scholar). Since the recognition of the DISC it has generally been considered that activation of the extrinsic pathway occurred almost exclusively at the plasma membrane. Although endocytosis of TNF and its associated receptors has long been recognized (8Mosselmans R. Hepburn A. Dumont J.E. Fiers W. Galand P. J. Immunol. 1988; 141: 3096-3100PubMed Google Scholar, 9Tsujimoto M. Yip Y.K. Vilcek J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7626-7630Crossref PubMed Scopus (354) Google Scholar, 10Watanabe N. Kuriyama H. Sone H. Neda H. Yamauchi N. Maeda M. Niitsu Y. J. Biol. Chem. 1988; 263: 10262-10266Abstract Full Text PDF PubMed Google Scholar), it was not considered to be important in signaling to death. However, independent studies in our laboratory and others recently demonstrated that caspase-8 and FADD are not recruited to a TNF-induced plasma membrane-bound receptor signaling complex but, instead, are activated elsewhere within the cell (11Harper N. Hughes M. MacFarlane M. Cohen G.M. J. Biol. Chem. 2003; 278: 25534-25541Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 12Micheau O. Tschopp J. Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (1966) Google Scholar, 13Schneider-Brachert W. Tchikov O. M. P. A. J. D. M. S. J. PubMed Scopus Google Scholar, W. Tchikov J. M. S. J. M. O. M. D. R. D. S. Full Text Full Text PDF PubMed Scopus Google Scholar). has been that an complex TNF, death TNF factor and is that after or to their death to FADD, which in recruits and caspase-8 O. Tschopp J. Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (1966) Google Scholar). Induction of apoptosis by CD95 to by two main in type I cells it formation of of DISC by receptor internalization and not require a mitochondrial or pathway and in type which form a DISC and and require with mitochondria A. P. S. Peter M.E. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, Peter M.E. Immunol. 2003; PubMed Scopus Google Scholar). a requirement for receptor internalization for CD95 DISC and caspase activation in type I cells has also been Tchikov R. S. Peter M.E. EMBO J. PubMed Scopus Google Scholar). was that recruitment of DISC occurred primarily after internalization of the receptor an and also that of internalization CD95 DISC formation and apoptosis in type I cells Tchikov R. S. Peter M.E. EMBO J. PubMed Scopus Google Scholar). there to be a for internalization of TNF-R1 and CD95 in apoptosis to a for internalization of TRAIL and its receptors in TRAIL-induced apoptosis. show that TRAIL is rapidly primarily by clathrin-dependent endocytosis but also by clathrin-independent endocytosis. internalization of TRAIL or its receptors is not required to a apoptotic signal, unlike for TNF-R1 and CD95 W. Tchikov J. M. S. J. M. O. M. D. R. D. S. Full Text Full Text PDF PubMed Scopus Google Scholar, Tchikov R. S. Peter M.E. EMBO J. PubMed Scopus Google Scholar). the TRAIL DISC is in the of internalization and is to the apoptotic after to TRAIL. Cell and BJAB cell and BJAB cells J.C. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google by A. of BJAB cells in with and cells wild type or a dominant negative form of of a H. J. Cell Biol. PubMed Scopus Google Scholar, H. M. S. H. 1995; PubMed Scopus Google by R. of with from S. cells in of and with and dominant negative and a AP180 from J. of of the within these by and from to FADD dynamin and AP180 from was from and used as S.B. M. MacFarlane M. Cohen G.M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). and TRAIL was and as M. M. Cohen G.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and tetramethylrhodamine ethyl from and cell from sucrose was from caspase benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl was from was and as MacFarlane M. J. Cohen G.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of TRAIL and TRAIL, was with and by the of by for in the was with a of ligand was was in the of compared with TRAIL to induce apoptosis in BJAB cells not for TRAIL and by of TRAIL and cells in at 4 in the or of TRAIL or in the of ligand for the rapidly to inhibit endocytosis and at 4 in 1 1 cell ligand was by in for in and in of ligand internalization by a in the of and with and in in the of and cells Internalization of TRAIL and in cells was as that after of cell ligand and cells and Induction of dynamin or dominant negative dynamin was by not of Internalization by cells to 4 for 1 and then to TRAIL for and with at 4 for and in or TRAIL was to at for the to cells to and in for at with the and a with and with and cells wild type or the dominant negative and internalization of TRAIL or with was in the as BJAB cells. cells after with and the plasma was to of Cell and Internalization cells and with 1 and 1 Cell surface proteins with to the with and in TRAIL and for to ligand with to ligand and in at 4 or at for the and to with cells in 1 and 1 and for to cell surface was by cells with Cell in DISC and proteins with protein was used to Inhibition of by inhibit receptor BJAB cells for in sucrose and cells in sucrose and for at to sucrose was its to receptor internalization levels of apoptosis within the of cell in BJAB cells. of in BJAB cells was by of mitochondrial potential BJAB cells with at for apoptosis by cells with and at for with and by a and the of DISC cells with TRAIL for to at or cells for with TRAIL for and or to for to cells and TRAIL and the associated proteins and DISC proteins by as N. A. Cohen G.M. MacFarlane M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of BJAB by cells in of and 1 a for of cells was of and in a at for 4 was by the cell and of TRAIL or or of by at a of in for cells and in BJAB cells used at a of TRAIL was used at a of TRAIL, with 1 soluble TRAIL at 4 for with the of cells. of TRAIL used to was by of TRAIL the of soluble TRAIL. of apoptosis to 1 TRAIL was to be to soluble TRAIL the of these was by of Internalization of TRAIL in BJAB of TRAIL was in BJAB cells to TRAIL. with TRAIL to endocytosis or at for to the cell ligand was by and TRAIL was by the to in a internalization of TRAIL with at and for to of TRAIL as as in of TRAIL the of the and the of TRAIL-R2 in the endocytosis of TRAIL, compared the levels of TRAIL internalization in TRAIL-R2 BJAB cells J.C. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of as a of TRAIL-R1 in the BJAB cells J.C. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google not of TRAIL was but not in compared with wild type BJAB of was and with the that internalization of TRAIL is mediated TRAIL-R1 and of at °C, used as a was almost by hyperosmotic sucrose sucrose is known to endocytosis of ligands and receptors J.E. J. Cell Biol. PubMed Scopus Google Scholar, D. N. M. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of TRAIL was by Internalization of TRAIL was also by in wild type BJAB cells at 4 a of TRAIL the cell and TRAIL to be the cells to °C, internalization of TRAIL was at Internalization was at with of and was by with in S. D. R. K. M. M. M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Internalization for Cell the of TRAIL internalization in the of soluble TRAIL was to a by in at 4 and in the of BJAB cells to the in the or of apoptosis and soluble TRAIL was our demonstrate that ligand internalization is not an requirement to an apoptotic in to TRAIL, but do not the that the is as of the death internalization of TRAIL was not required for cell to the of of the cell to ligand in to induce apoptosis. BJAB cells in the of TRAIL for by to a in the of TRAIL and for a of 4 to TRAIL for was to induce for was to induce levels of apoptosis these demonstrate that TRAIL not be to induce and ligand and receptor that within the are to apoptosis. and the of mitochondrial potential as a of cell death by apoptosis. that was a of TRAIL-induced apoptosis in BJAB the of and it with the in and the of a used of apoptosis. in with the in and the of the was compared with the of or in with at a of apoptosis. of a caspase the of cell death by these these demonstrate that TRAIL a of apoptosis in BJAB and the of be by of TRAIL-induced apoptosis of BJAB cells BJAB cells to TRAIL for the and cell death was by the of cells of mitochondrial potential with TMRE, with or by also with TRAIL for 4 in the of a caspase results shown are the from in a Internalization after Ligation by internalization of TRAIL was not to induce it was that internalization of surface was receptor cell surface proteins with a and cells with TRAIL for internalization for at °C, receptors by as internalization of of TRAIL-R1 and was in the of ligand with levels of receptor endocytosis Varfolomeev E.E. K. J. D. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). with TRAIL a of internalization of TRAIL-R1 and internalization of these receptors was associated with recruitment of FADD and caspase-8 Caspase-8 was also to its forms and forms and not of BJAB cells with hyperosmotic sucrose in an almost of internalization of TRAIL-R1 and of the of receptors, FADD and of caspase-8 Internalization for TRAIL-induced the of receptor internalization in TRAIL-induced BJAB cells in normal or hyperosmotic in the of TRAIL and apoptosis was by of a in apoptosis was in cells to TRAIL hyperosmotic 4 hyperosmotic did not induce apoptosis with an results suggest that receptor internalization is not required for TRAIL-induced apoptosis and suggest that internalization the apoptotic hyperosmotic induce apoptosis by activation of the intrinsic or extrinsic pathway A. M. S. PubMed Scopus Google Scholar, R. D. J. 2003; 17: PubMed Scopus Google Scholar). hyperosmotic was to TRAIL or BJAB cells with a of selectively death apoptosis apoptosis induced by perturbation of mitochondria and activation of the intrinsic pathway MacFarlane M. J. Cohen G.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the apoptosis after of cells in hyperosmotic with TRAIL that hyperosmotic was the cells to TRAIL-induced apoptosis. of TRAIL-induced caspase an in caspase-8 activation after 1 in cells with hyperosmotic which with an in 1 and of apoptosis was in cells for 4 in hyperosmotic in with an of of caspase-8 or and 4 in hyperosmotic TRAIL induced almost of caspase-8 and and the of caspase-8 was by the of a of the TRAIL-induced of caspase-8 and was almost by as by the of almost the caspase-8 and Although hyperosmotic TRAIL-R1 and of apoptosis and activation of not that TRAIL receptor internalization is not for cell death signal in TRAIL-induced apoptosis. Internalization for DISC our that internalization of TRAIL-R1 and was not required for TRAIL-induced to the of receptor internalization in DISC BJAB cells with TRAIL cells at 4 to the cells for internalization and to or at for the then and used to and DISC proteins by of cells to TRAIL at in formation of a DISC within and of TRAIL-R1 and TRAIL-R2 with the recruitment of FADD and procaspase-8 at two caspase-8 most the two and of TRAIL-R1 and TRAIL-R2 and recruitment of FADD and caspase-8 at and for but a at was to its and a of its was also and of TRAIL-R1 and to TRAIL, FADD and caspase-8 associated with the receptors because from the to the internalization of the receptor complex a cellular the results in of the in the DISC BJAB cells with TRAIL at 4 by TRAIL and then the cells to at at 4 TRAIL to TRAIL-R1 and TRAIL-R2 by recruitment of FADD and the of caspase-8 to the DISC to °C, the levels of FADD in the levels of TRAIL-R1 and TRAIL-R2 to of the ligand with TRAIL-R1 and TRAIL-R2 at the A. S. S. M.P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). caspase-8 was its forms with a of the the of caspase was almost with a corresponding in the of and by the was its and results demonstrate that caspase-8 recruited to the DISC was rapidly to its and forms and did not the that caspase-8 that associated with the DISC at was recruited the of the of the finding of FADD and caspase-8 recruitment to the DISC at 4 a of TRAIL-R1 and TRAIL-R2 that was by the recruitment of FADD and caspase-8 of to TRAIL at 4 °C, a of of TRAIL-R1 and TRAIL-R2 was by recruitment of of FADD and caspase-8 to the DISC of recruitment of DISC proteins levels and did not within the of the a of caspase-8 was internalization of receptors is at 4 °C, these demonstrate that internalization of TRAIL its receptors is not required for the formation of a DISC for TRAIL-induced Cell was in it was that TRAIL-R2 was by endocytosis Varfolomeev E.E. K. J. D. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the of clathrin-dependent internalization in the of apoptosis. BJAB cells with a dominant negative the of AP180 which with the recruitment and of at the plasma Y. A. PubMed Scopus Google Scholar). of TRAIL-induced apoptosis and ligand internalization in the cell cells with a protein of the of was by overexpression of dominant negative or these internalization of TRAIL was also by overexpression of or dominant negative dynamin at after to cell death that occurred at after of dominant negative or not of cells with did not the of BJAB cells to TRAIL-induced apoptosis that ligand internalization is not required for TRAIL-induced apoptosis in BJAB cells. Blockade of TRAIL-induced in of endocytosis of that inhibit endocytosis did not to inhibit TRAIL-induced apoptosis in BJAB to in a cellular with of endocytosis. used a of cells a dominant negative the of a H. J. Cell Biol. PubMed Scopus Google Scholar, H. M. S. H. 1995; PubMed Scopus Google Scholar). cells dynamin at but not at 4 °C, internalization of in cells the dynamin was at of was in the dominant negative compared with cells a internalization results demonstrated that endocytosis was in the that the wild type and cells as shown the of the dominant negative dynamin TRAIL internalization internalization of TRAIL was or cells at 4 TRAIL was within of to in cells dynamin with TRAIL at the plasma TRAIL was also in the cells the dynamin TRAIL to at the cell that TRAIL was or a clathrin-independent in the cells Although a in TRAIL in the dominant negative cells compared with cells was TRAIL to be our results the that TRAIL be by but pathway is it also be by clathrin-independent endocytosis. TRAIL internalization was in cells the dynamin the of clathrin-dependent endocytosis in TRAIL-induced apoptosis. was by the of cells by levels of apoptosis in the wild type and dynamin cells 1 and for 4 of cells or dominant negative dynamin to TRAIL the levels of apoptosis and TRAIL induced levels of apoptosis in of that clathrin-dependent endocytosis is not required for TRAIL-induced apoptosis. Our studies have a requirement for and internalization in TRAIL-induced apoptosis a of We demonstrate that there is requirement for endocytosis of ligand or receptor TRAIL-induced apoptosis. show that TRAIL-induced recruitment of FADD and procaspase-8 to its receptors to form the TRAIL DISC occurs at 4 °C, a endocytosis is demonstrate that of the TRAIL DISC occurs in the of of endocytosis of TRAIL and its cognate receptors hyperosmotic and to apoptotic signaling TRAIL-induced apoptosis did not induce apoptosis of clathrin-dependent endocytosis by overexpression of a dominant negative form of dynamin and of AP180 in two cell and and did not with the of TRAIL to apoptosis. was our finding in BJAB cells that the of the TRAIL DISC with associated FADD and caspase-8 occurred at 4 which is endocytosis is Although the of cells as type I or their requirement or not for the of a mitochondrial has been primarily used in their to CD95 A. P. S. Peter M.E. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, Peter M.E. Immunol. 2003; PubMed Scopus Google Scholar), it has also recently been used to of cells to TRAIL-induced apoptosis N. 2002; PubMed Scopus Google Scholar, J. K. S. 2005; PubMed Scopus Google Scholar). BJAB cells are type I cells in of TRAIL used in the studies the formation of the TRAIL DISC N. S. P. P. P. J. Tschopp J. Nat. Cell Biol. 2000; 2: PubMed Scopus Google Scholar, F.C. A. P. A. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. Krammer P.H. H. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). levels of FADD and procaspase-8 recruited to TRAIL receptors at 4 as as after the of ligand to BJAB with of TRAIL DISC after ligand formation of the TRAIL DISC in BJAB cells occurs the of endocytosis or of these findings a in the requirement for internalization for DISC TRAIL receptor and signaling Tchikov R. S. Peter M.E. EMBO J. PubMed Scopus Google Scholar, Varfolomeev E.E. K. J. D. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the requirement for endocytosis in but not TRAIL-induced is to the of CD95 but not TRAIL receptors to studies A. M. M. I. R. J. Immunol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Nat. Immunol. 5: PubMed Scopus Google Scholar). of the TRAIL DISC at 4 the as to a of FADD with TRAIL receptors in the of and of the cell surface receptors with TRAIL induces a that results in much of FADD to TRAIL-R1 and FADD caspase-8 are in the DISC at 4 of receptors and signaling in the of ligand has been for of with type I and receptors K. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). studies have a requirement for receptor internalization in death apoptosis for TNF-R1 and W. Tchikov J. M. S. J. M. O. M. D. R. D. S. Full Text Full Text PDF PubMed Scopus Google Scholar, Tchikov R. S. Peter M.E. EMBO J. PubMed Scopus Google Scholar). clathrin-dependent endocytosis was required for and of a CD95 DISC within an in type I cells Tchikov R. S. Peter M.E. EMBO J. PubMed Scopus Google Scholar). CD95L and TRAIL induce of a adaptor which formation and at the plasma Varfolomeev E.E. K. J. D. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). and M. with TRAIL clathrin-dependent endocytosis Varfolomeev E.E. K. J. D. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the that to endocytosis is an in of TRAIL in dominant negative cells and BJAB cells with dominant negative dynamin or and with the that pathways, such as the recently pathway, to TRAIL endocytosis Nat. Cell Biol. PubMed Scopus Google Scholar). overexpression of or for or levels not the that be after of internalization is that endocytosis to the internalization of TRAIL receptors, at in BJAB as these cells do not TRAIL and its receptors and rapidly in the type I cell BJAB 1 and with internalization of ligand and its receptors as as after TRAIL Although neither nor internalization clathrin-dependent endocytosis to be required for TRAIL-induced ligand receptor internalization in signaling such as protein or activation H. D. D. M. D. A. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of studies have shown that TRAIL formation of a signaling complex after of the TRAIL DISC H. D. D. M. D. A. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). to the DISC FADD and caspase-8, signaling platform also RIP, TNF factor and which are in the activation of protein and studies are to the of these TRAIL signaling and endocytosis is required for their Although and have shown that TRAIL and its receptors are the of TRAIL receptors that TRAIL-R1 and TRAIL-R2 the plasma and of cells A.V. P. J. Immunol. 2000; PubMed Scopus Google Scholar). TRAIL induced an in cell surface TRAIL-R1 and of these receptors to the cell surface also occurred A.V. P. J. Immunol. 2000; PubMed Scopus Google Scholar). it is that of TRAIL receptors to the plasma as a to or TRAIL-induced in cells that have a of to cell surface TRAIL to endocytosis of TRAIL and its receptors 1 and that TRAIL-induced and and apoptosis induced apoptosis and of the apoptosis after of cells with TRAIL in the of compared with normal that hyperosmotic was the cells to TRAIL-induced apoptosis Although do not the that such as activation of family or protein or of in the by which cells to TRAIL-induced our the that of endocytosis to apoptosis by formation of a cell surface TRAIL DISC. our studies show that TRAIL and its receptors are rapidly clathrin-dependent but also by clathrin-independent in are not required for apoptosis induced by TRAIL. is in to death receptor ligands TNF and formation of the TRAIL DISC in BJAB type I cells in the of receptor with